Fluorescent calcium indicators that are ratiometric and emit in the red spectrum

Abstract

A new generation of fluorescent ion indicators for measuring cytosolic concentrations of calcium. These new indicators emit in the red portion of the visible spectrum and are ratiometric. The ratiometry consists of shifting in the emission upon binding calcium when exciting with an argon laser at 488 nm. These indicators also show marked increases in red fluorescence when excited with visible light at longer wavelengths. Like all calcium indicators, they comprise the usual chelating portion based on BAPTA (Bis Amino Phenyl Tetraacetic Acid) and a fluorophore portion, either a seminaphthofluorescein / seminaphthorhodamine or dinaphthofluorescein, which enable ratiometry. Unlike other naphthofluoresceins, the pKa has been adjusted on the naphtho moiety to make it suitable for biological calcium measurements. The invention relates to the structures of the indicators and to the method of synthesizing these indicators.

Description

RELATED APPLICATIONS[0001]This application is related to U.S. Provisional Patent Application No. 61 / 252,654 filed Oct. 17, 2010 by applicants, and claims the priority date of that application.BACKGROUND[0002]1. Field of Invention[0003]This application relates to the design and synthesis of long wavelength fluorescent ion indicators.[0004]2. Prior Art[0005]The most popular fluorescent calcium indicators that are ratiometric include Fura-2 (excitation shifting) and Indo-1 (emission shifting)i. Ratiometry has the unique advantage of eliminating the variable effects in calcium measurement such as indicator concentration, degree of cell loading, photobleaching, detector sensitivity, cell thickness, and optical path. It is essentially an ideal property that allows for calibration and quantitative measurements. However, these popular ratiometric dyes operate in ultraviolet light. UV optics are expensive and hence have limited the general use of these dyes. The high energy UV light can also...

AI Technical Summary

Benefits of technology

[0011]The pKa of the seminaphthofuorescein and seminaphthorhodamine is reduced by halogenation of the naphthol moiety with fluorines and chlorines. Thus, the pKa of the nonhalogenated seminaphthofluorescein is reduced from 7.8 to 5.5 via di-orthohalogenation, making the dies unresponsive to small pH changes in the cell.

[0020]The third of these compounds has a unique chelating backbone. The BAPTA has an extra carboxylic function ortho to the fluorophore. The dye conjugated to this BAPTA most closely resembles 6-aminoseminaphthorhodamine for an enhanced quantum yield. The naphthols have chlorine or fluorine substitution to reduce the pKa from greater than 8 to 5.5. The emission maximum is in the red at 650 nm.

Problems solved by technology

UV optics are expensive and hence have limited the general use of these dyes.

The high energy UV light can also be harmful to the cell.

The intrinsic fluorescence due to nucleotides and some amino acids when exciting with UV light also causes interference during measurement, although ratiometry does attenuate this problem.

The first, BTCiii, has a very high dissociation constant Kd that does not allow for measurement of small changes in cytosolic concentrations (nanomolar) of calcium.

The second, Fura-Rediv, has a low quantum yield in water due to its hydrophobicity and therefore has not found much use in cellular studies.

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